International Journal of Environmental Analytical

Chemistry

ISSN: 0306-7319 (Print) 1029-0397 (Online) Journal homepage: https://www.tandfonline.com/loi/geac20

Automatic flow system for evaluation of

polystyrene-divinylbenzene sorbents applied to
preconcentration of phenolic pollutants

Hugo M. Oliveira , Marcela A. Segundo & José L.F.C. Lima

To cite this article: Hugo M. Oliveira , Marcela A. Segundo & José L.F.C. Lima (2011) Automatic
flow system for evaluation of polystyrene-divinylbenzene sorbents applied to preconcentration of
phenolic pollutants, International Journal of Environmental Analytical Chemistry, 91:9, 884-899,
DOI: 10.1080/03067310903276316

To link to this article: https://doi.org/10.1080/03067310903276316

Published online: 06 Jul 2011.

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Intern. J. Environ. Anal. Chem.
Vol. 91, No. 9, 10 August 2011, 884–899

Automatic flow system for evaluation of polystyrene-divinylbenzene

sorbents applied to preconcentration of phenolic pollutants
Hugo M. Oliveira, Marcela A. Segundo* and José L.F.C. Lima

REQUIMTE, Serviço de Quı´mica-Fı´sica, Faculdade de Farmácia,

Universidade do Porto, Rua Anı´bal Cunha, 164, 4099-030 Porto, Portugal
(Received 25 June 2009; final version received 17 August 2009)

In this work, an evaluation of commercially available polystyrene-divinylbenzene

sorbents for solid-phase extraction (SPE) of eleven phenolic compounds is
intended. Considering the particle size and cross-linking degree, Amberlite
XAD-4 (commercial or grounded), Macronet MN-200 and Lichrolut EN were
tested. The SPE protocol was performed by an automatic system, providing
repeatable experimental conditions for assessment of sorbent capacity, break-
through volume and enrichment factor (EF). A positive correlation between EF
and log Kow was found for Amberlite XAD-4 while a negative correlation was
observed between EF and molecular weight of analyte for Macronet MN-200
and for Lichrolut EN. This indicates a prevalence of hydrophobic interactions
or molecular exclusion depending upon the polymer cross-linking degree.
Despite the similar repeatability (RSD 5 4.7%, n 4 6) and recovery values
attained (97.6–102.7%, using 50 mL of sample) for all sorbents, Lichrolut EN is
the best choice for analytical application as higher EF and lower LOD values
(between 18 and 207 ng) were attained for this sorbent.
Keywords: automation; multisyringe flow injection analysis; phenolic pollutants;
polystyrene-divinylbenzene; solid-phase extraction

1. Introduction
Phenolic compounds are ubiquitous pollutants generated in the production of plastics,
dyes, drugs, pesticides, paper and also in the petrochemical industry [1]. Due to the intense
and unpleasant organoleptic properties and toxicity at low concentration levels (mg L1),
these compounds have been included in the United States Environmental Protection
Agency (EPA) list of priority pollutants [2]. The determination of EPA phenolic pollutants
in water samples from different sources is usually performed by separation techniques such
as GC-MS [3], LC coupled to ultra-violet [4] or electrochemical detection [5]. Although
GC-MS provides higher sensitivity than LC-UV, the derivatisation of the analytes prior
to chromatographic run is usually unavoidable. Furthermore, a previous sample
preparation step comprising the preconcentration of the target analytes and matrix
removal is always required for both chromatographic methodologies.
In this context, solid-phase extraction (SPE) is a suitable technique for performing
the enrichment of water samples [6] because it requires low amounts of organic solvents
and allows the processing of large sample volumes. Three categories of sorbents, based on

*Corresponding author. Email: msegundo@ff.up.pt

ISSN 0306–7319 print/ISSN 1029–0397 online

ß 2011 Taylor & Francis
DOI: 10.1080/03067310903276316
http://www.informaworld.com
 International Journal of Environmental Analytical Chemistry 885

silica, carbon or polymeric materials, have been used to extract phenolic compounds
from water samples by a reversed-phase mechanism [7]. Besides the limited pH working
range, silica sorbents have the inconvenience of providing low recoveries for the more
polar compounds [8]. On the other hand, when carbon sorbents were used, poor recoveries
were achieved due to difficulties in removing the trapped compounds from the sorbent
surface [9]. These sorbents also have poor mechanical stability, and band broadening
was observed in the chromatograms [9,10]. In contrast, polymeric materials do not have
limitations in the pH working range, have good mechanical properties and provide
quantitative recovery of phenolic compounds [11].
Generally, polymeric sorbents comprise a polystyrene-divinylbenzene (PS-DVB)
hydrophobic structure. Different particle size and cross-linking degree are available,
depending on the supplier. Hence, the objective of this work was to evaluate commercially
available polymeric sorbents concerning their performance for SPE of EPA priority
pollutants prior to their determination. Three adsorbents were chosen: Amberlite XAD-4,
Lichrolut EN and Macronet MN-200. These were chosen considering the different
particle size and the cross-linking grade, which results in different surface areas. Amberlite
XAD-4 was used in the commercial form (300–850 mm) and as the grounded product
(125–250 mm) [12]. The commercial product has a cross-linking degree lower than 16% [13]
and a surface area of 750 m2 g1. Macronet MN-200 is classified as a hyper cross-linking
degree sorbent with particle size in the range 300–1200 mm and a surface area of
1000 m2 g1 [14]. Finally, Lichrolut EN particle distribution is in the range 40–120 mm,
and a surface area of 1200 m2 was achieved for 1 g of this PS-DVB sorbent with high
cross-linking degree [15]. Amberlite XAD-4 and Lichrolut EN are currently applied to
SPE of phenolic compounds [16,17]. Although generally used for wastewater treatment
through sorption and removal of phenolics from waste waters, Macronet MN-200 has
only once been applied for analytical purposes, to the best of our knowledge [18].
In order to attain repeatable experimental conditions, the comparison was established
using a multisyringe flow injection analysis (MSFIA) system [19] to perform the
concentration and elution steps of the SPE protocol in an automatic fashion. This type
of automatic flow system enables the assembly of a flow network, where solutions from the
different syringes can be either delivered to the flow system or returned to its own vessel,
without interfering with the other channels [20]. Several elements may be incorporated
into the flow network, including solid-phase reactors or columns [21,22]. In the present
work, the outlet of the SPE column was connected to the LC sample loop [23], which was
automatically filled with eluate before each chromatographic determination, assuring
repeatable conditions.

2. Experimental
2.1 Reagents and solutions
All chemicals used were of analytical-reagent grade and used with no further purification.
A MilliQ system was used to obtain ultra-pure water (resistivity 4 18 M
cm), used for
the preparation of all aqueous solution. Methanol HPLC grade (Merck, Darmstadt,
Germany) was used for the preparation of all methanolic solutions and the same solvent
was also used as eluent. The sorbents Amberlite XAD-4, Macronet MN-200 and Lichrolut
EN were obtained from Fluka (Buchs, Switzerland), Purolite (Brasov, Romania) and
886 H.M. Oliveira et al.

Table 1. Detection wavelength (), molecular weight, partitioning coefficients octanol/water

(log Kow) and dissociation constants (pKa) of EPA phenolic priority pollutants [25,26].

Phenolic compound  (nm) MW (u) log Kow pKa

2,4-Dinitrophenol (24DNP) 360 184.1 1.53 4.09

2-Methyl-4,6-dinitrophenol (46DNOC) 375 198.1 2.12 4.34
Phenol (P) 215 94.1 1.50 9.99
4-Nitrophenol (4NP) 315 139.1 1.90 7.16
2-Chlorophenol (2CP) 195 128.6 2.15 8.55
2-Nitrophenol (2NP) 210 139.1 1.78 7.21
2,4-Dimethylphenol (24DMP) 195 122.7 2.42 10.6
4-Chloro-3-methylphenol (4C3MP) 195 142.6 3.10 9.55
2,4-Dicholorophenol (24DCP) 200 163.0 2.08 7.85
Pentachlorophenol (PCP) 220 266.3 5.01 4.93
2,4,6-Trichlorophenol (246TCP) 200 197.5 3.69 7.42

Merck (Darmstadt, Germany), respectively. Amberlite XAD-4 with particle size in the
range 125–250 mm was prepared as described in a previous work [24].
The phenolic compounds listed in Table 1 were obtained from Sigma-Aldrich
(St. Louis, MO, USA). A stock solution of each compound was prepared by weighing
100 mg of the respective compound followed by dissolution in methanol in order to obtain
a final concentration of 1000 mg L1. Stock solutions were rigorous diluted in methanol
in order to obtain standard solutions of each individual compound or mixtures of them.
For the standard solutions submitted to the preconcentration step, the dilution was
performed by using 10 mmol L1 aqueous HCl as solvent to match the pH of water
samples.
The aqueous component of the mobile phase for the chromatographic method was
a 50 mmol L1 sodium dihydrogen phosphate (Fluka) solution with pH adjusted to 5.25
using a sodium hydroxide solution (6 mol L1). The organic component of the mobile
phase was acetonitrile HPLC grade, obtained from Merck. The mobile phase was prepared
from a 36:64 solution of the organic and aqueous components, filtered through a 0.45 mm
porous membrane and degassed by ultrasounds before use.

2.2 Chromatographic determination of phenolics

The chromatographic method was performed by using a Merck/Hitachi LaChrom 7000
series (Hitachi Ltd., Tokio, Japan) equipped with a pump (L-7100), a diode array detector
(L-7455) and an interface (D-7000). The monolithic analytical column used for the
reversed-phase separation of the studied compounds was a Chromolith RP-18e
(100  4.6 mm id) coupled to a pre-column (5  4.6 mm id) of the same material
(Merck). The interface between the automatic SPE system and the chromatograph was
a Rheodyne 7725i high pressure manual injector (Rheodyne, Rohnert Park, CA, USA),
containing a loop with an internal volume of 20 mL. This loop was filled automatically
at the end of each SPE cycle and its content was injected into the HPLC system by the
same device.
The method used in this work was proposed by Cledera-Castro et al. [25], allowing
the isocratic separation of eleven EPA phenolic priority pollutants in less than
 International Journal of Environmental Analytical Chemistry 887

0.450
5

Absorbance (AU)
0.300
7
8
9
0.150
12 4
3 6 10 11

0.000
0.00 0.50 1.00 1.50 2.00 2.50 3.00
Time (min)

Figure 1. Typical chromatogram obtained under the conditions described in the text by direct
injection of the compounds. Concentration of each phenolic compound: 10 mg mL1. Peak labels:
(1) 24DNP, (2) 46DNOC, (3) P, (4) 4NP, (5) 2CP, (6) 2NP, (7) 24DMP, (8) 4C3MP, (9) 24DCP,
(10) PCP, (11) 246TCP. Mobile phase; acetonitrile : phosphate buffer (36 : 64 vol.); flow rate,
4.00 mL min1.

three minutes (Figure 1). The only modification to the original method was the
temperature. Room temperature (approximately 23 C) was adopted because no significant
differences in the chromatogram were observed when this temperature was compared
with the original value of 36 C.

2.3 Flow injection apparatus

The manipulation of solutions inside the flow conduits of the automatic SPE system
was performed by a BU4S multisyringe burette (Crison Instruments, Alella, Spain)
equipped with a syringe of 10 mL in position 2 and a syringe of 5 mL in position 3 and,
a Minipuls 3 peristaltic pump (Gilson, Villiers-le-Bel, France) equipped with poly-
vinylchloride pumping tubes. A three-way commutation valve (NResearch, Caldwell, NJ,
USA) connected to the head of each syringe of the multisyringe module allowed the
optional coupling to the manifold lines or to the solution vessel. An array of four extra
commutation valves controlled by the same module completed the basis of the flow
network of the present system. The commutation valves options were codified in on/off
lines. For the valves placed at the head of the multisyringe the ‘on’ line indicated
the communication of the solution to the flow network whereas the ‘off’ line was assigned
to the solution reservoir.
The control of the propulsion devices was performed by a personal computer running
lab-made software written in QuickBasic 4.5 (Microsoft, Redmond, WA, USA). Two
different hardware channels were used. The multisyringe module control (number of
steps, the direction of piston displacement and the position of the commutation valves)
was performed through a serial port and the peristaltic pump control (flow direction
and rotation speed) was mediated by an interface card (PCL-711, Advantech, Taipei,
Taiwan).
888 H.M. Oliveira et al.

(a)

St
PP

V6 V8 W
S1/V1

W
C S2/V2 EC

V7 V5 L1
EL S3/V3

S4/V4 IV
MS 1 6
CP
HP 2 5

3 4

MCC
W

(b)
MSFIA

1 6
CP
HP 2 5
W
3 4
MCC

Figure 2. (a) MSFIA-SPE manifold for the automatic preconcentration of phenolic compounds,
connected to the chromatographic system through the injection valve, represented on ‘load’ position.
(b) Schematic representation of the injection valve configuration on ‘inject’ position. MS,
muiltisyringe; Si, syringe; Vi, commutation valves; EC, SPE column; IV, injection valve; PP,
peristaltic pump; W, waste; L1, connection tubing (300  0.8 mm id); S2, syringe 10 mL; S3, syringe
5 mL; C, carrier (HCl 10 mmol L1); EL, eluent (methanol); St, sample or standard solution;
HP, high-pressure pump; MC monolithic chromatographic column; MSFIA, preconcentration
flow system; CP, closed port. In the commutation valves, the position ‘on’ is represented by a solid
line while the position ‘off’ is represented by a dotted line. The needle port and connection to waste
in IV are also represented by dotted lines.

2.4 Manifold and automatic SPE procedure

The automatic flow injection system components were arranged as shown schematically
in Figure 2. PTFE tubing with 0.8 mm id (Omnifit, Cambridge, UK) was used for all
connections. The different sorbents used in this work were packed in two different
columns. For trapping Amberlite XAD-4 commercial product (213 mg), Amberlite XAD-4
 International Journal of Environmental Analytical Chemistry 889

grounded product (194 mg) and Macronet MN-200 (188 mg), a stainless steel column
20 mm of length and 7 mm of internal diameter was used. A polyetheretherketone (PEEK)
device presenting the same tubular configuration with 24 mm length and 3 mm of internal
diameter was used for packing Lichrolut EN (87 mg). In both cases, the sorbents were
trapped inside the columns by using polypropylene filter disks supplied by MoBiTec
(Goettingen, Germany) with a pore diameter of 35 mm (stainless steel column) or 10 mm
(PEEK column).
The complete protocol sequence for the automatic SPE procedure, including nine
steps, is described in Table 2. The first command comprises the system cleaning (including
the extraction column) from the previous cycle with 3500 mL of methanol followed
by the sorbent bed conditioning with 2500 ml of HCl 10 mmol L1 (step 2). After this,
the peristaltic pump was activated at a flow rate of 4 mL min1, and a variable volume
of sample was loaded into the extraction column (step 3). Afterwards, the syringes were
refilled with the volume of methanol and HCl solution necessary for the next two steps
(step 4). Subsequently, 2500 mL of HCl solution were propelled through the sample
channels and the extraction column in order to remove residues of the compounds (step 5).
After the commutation of the appropriate valves, a variable volume of methanol
(dependent of the sorbent used) was sent in the direction of the sorbent bed to elute the
analytes (step 6). During this step the injection valve loop was filled and 20 mL of the eluate
were injected into the chromatographic system by rotating the HPLC injection valve
to the ‘inject’ position (step 7), beginning the chromatographic run. At last, the syringes
were refilled (step 8) and the injection valve was turned to the ‘load’ position (step 9).
Thereafter, the system was ready for the next SPE cycle.

3. Results and discussion

3.1 Design of the automatic SPE method
The SPE automatic manifold was based on a multisyringe burette that allowed a high
precision in controlling the flow rates and volumes of solvents/solutions used for sorbent
elution and conditioning. Furthermore, the assembling of a peristaltic pump into the
manifold for propelling the sample through the extraction column enabled drastic
reduction of the time necessary for the sampling step. Otherwise, the sample would be
propelled by one of the burette syringes, but it would require several washing steps to
prevent carryover between samples.
The inclusion of the extraction column between two commutation valves (V5 and V7)
allowed that the operations of loading and elution were performed in opposite ways,
avoiding the possible compaction of the sorbent particles and the consequent creation
of back pressure which could result in the modification of the flow rate or in the column
clogging. The commutation valve V8 provided an alternative flow path for the sample
exchange without contamination of the extraction column.
The performance of the different sorbents was assessed by considering the
sorbent capacity, the breakthrough volume and the enrichment factor obtained after the
preconcentration step. The evaluation of the elution volume, breakthrough volume
and the sorbent capacity was performed using P and 246TCP as model compounds. These
compounds were chosen because they represented the extreme zones of polarity and
molecular weight (Table 1) and they were also located in the extreme zones of retention
time in the chromatographic run (Figure 1).

Table 2. Procedure for the automatic solid-phase extraction of phenolic compounds prior to its chromatographic analysis.
Step Instrumentation
1 Multisyringe piston pump
2 Multisyringe piston pump
3 Peristaltic pump
4 Multisyringe piston pump
5 Multisyringe piston pump
6 Multisyringe piston pump
7 HPLC injection valve
8 Multisyringe piston pump
9 HPLC injection valve
890
Protocol Description
Dispense 3500 mL at 3 mL min1 with heads V2 off, V3 on, V5 on, Washing of sorbent bed and injection valve
V6 off, V7 on, V8 off, IV load loop with methanol
Dispense 2500 mL at 5 mL min1 with heads V2 on, V3 off, V5 off, Sorbent conditioning with HCl, pH 2.0
V6 on, V7 off, V8 off, IV load
Propel X mL at 4 mL min1 with heads V2 off, V3 off, V5 off, V6 off, Sample loading (variable volume up
V7 off, V8 off, IV load to 100 mL)
Pickup 3720–4350 mL at 15 mL min1 with heads V2 off, V3 off, Piston bar adjustment
V5 off, V6 off, V7 off, V8 off, IV load
Dispense 2500 mL at 5 mL min1 with heads V2 on, V3 off, V5 off, Matrix removal and tubing cleanup with HCl,
V6 on, V7 off, V8 off, IV load pH 2.0
H.M. Oliveira et al.
Dispense 610–925 mL at 1.5 mL min1 with heads V2 off, V3 on, Elution of the analytes using methanol
V5 on, V6 off, V7 on, V8 off, IV load
Rotate IV to inject position Start of chromatographic run
Pickup 9500 mL at 15 mL min1 with heads V2 off, V3 off, V5 off, Piston bar adjustment
V6 off, V7 off, V8 off, IV inject
Rotate IV to load position Connect IV loop to MSFIA-SPE system
 International Journal of Environmental Analytical Chemistry 891

The initial conditions for the operation of the automatic SPE system were fixed
according to previous work describing on-line coupling of the PS-DVB sorbents with
liquid chromatographic systems [26]. The flow rates used during the SPE cycle were chosen
considering the time of contact between the solutions (washing/conditioning solution,
sample and eluent) and the sorbent, but also the existence of back pressure during
the operation and the dispersion of the segment of eluate before filling the loop of the
chromatographic injection valve. For these reasons flow rates of 4 mL min1 for sample
loading, up to 5 mL min1 for sorbent system washing/conditioning and 1.5 mL min1 for
the elution step were used in all experiments.
The volumes used for system washing/conditioning were selected as the minimum
values that avoided ‘memory effect’ between samples and a correct conditioning of the
extraction column before the sample loading step. A volume of 3500 mL of methanol was
sent through the extraction column to perform the removal of all analytes which could be
present into the flow line and two portions of 2500 mL of HCl 10 mmol L1 were sent by S2
to the SPE device immediately before and after the sample loading in order to conditioning
the sorbent and for removing sample residues before elution, respectively.
The elution volume was fixed after obtaining the elution profile of the model
compounds. The experiment consisted in the extraction of 5 mg of P and 246TCP and
subsequent evaluation of the peak area obtained with different elution volumes in the
range 550–1250 mL. Similar elution profiles were obtained for both compounds in
each sorbent, indicating methanol as a suitable solvent for desorption of the analytes.
The volume for which the maximum peak areas were obtained varied from 610 mL
(for Lichrolut EN) to 925 mL (for Amberlite XAD-4 125–250 mm). These differences
were observed due to the difference of mass of sorbent used and the bed volume of the
extraction columns. The length of the connection tubing between the extraction column
and the chromatograph injection valve loop (L1, Figure 2) was the minimum possible
in order to minimise the dispersion and consequently the dilution of the segment of eluate.

3.2 Sorbent loading capacity

For each sorbent a calibration curve was established by extracting 5 mL of individual
standard solutions of P and 246TCP in the range 1–10 mg L1. The volume loaded was
selected in order to guarantee that no breakthrough occurs. In all cases, no deviations
in the linearity of the calibration curve were observed up to the maximum amount of P and
246TCP loaded, which corresponded to 35 mg of each compound for Lichrolut EN and
50 mg for the remaining sorbents. These results demonstrated that no overloading occurred
up to the maximum amounts of the compounds extracted. The amount of 50 mg of P and
246 TCP was not tested with Lichrolut EN due to the high value of peak area (maximum
peak height 4 1 AU) obtained for concentrations above 7 mg L1.

3.3 Breakthrough volume

The breakthrough volume corresponds to the largest sample volume that can be processed
without loss of analyte and for which recovery for all sample volumes lower than the
breakthrough volume will be 100% [27]. This value defines the maximum volume which
allows an exhaustive extraction of the analyte from the matrix. Two classical methods have
been described for the experimental measurement of the breakthrough volume [6,27].
892 H.M. Oliveira et al.

The frontal analysis method is based on the continuous feeding of the SPE column with
analyte, accompanied by continuous or discrete monitoring of the non-retained analyte
by detecting the UV signal at the outlet of the sorbent bed. Another common approach
to estimate the breakthrough volume is the preconcentration of different sample volumes,
each containing the same amount of analytes and then measuring the analytical signal
obtained after elution of the compounds. Both methods are time-consuming and may not
reflect the real working conditions, especially the concentration levels used when sample
analysis is performed. For these reasons, in the present work, a different experimental
approach based on the enrichment of different sample volumes with a constant
concentration of analyte is proposed, by taking advantage of the time based sampling
of the automatic flow system. Therefore, the loading volume was defined (and changed)
by computer control, after fixing the flow rate and the time during which the peristaltic
pump was activated (Table 2, step 3). A mass calibration curve was established by plotting
the peak area against the mass of analyte loaded into the column [28]. This experiment
consisted in the extraction of sample volumes between 1 and 100 mL of a standard
solution containing 500 mg L1 of the model compound. This concentration was chosen
in order to guarantee a maximum amount of compound that did not cause overloading
of the extraction column. In the calibration curve, when a deviation from the linearity
of the calibration curve was observed, it was considered that breakthrough occurred [29].
The results obtained for all sorbents (Table 3) are expressed as the volume range, defined
by the two experimental points between which breakthrough occurred, or as the maximum
volume loaded without breakthrough.
Due to the hydrophobic character of the sorbents used, the values of breakthrough
volume were lower for the more polar compound tested (P). For the sorbents with high
cross-linking degree, as Macronet MN-200 and Lichrolut EN, no breakthrough was
observed up to the maximum volume of 246TCP loaded (100 mL). The best performance
for P was achieved when Amberlite XAD-4 (grounded product) and Lichrolut EN were
used, with breakthrough volumes above 75 mL. Considering the results obtained for both
model compounds, the highest breakthrough volumes were achieved for Lichrolut EN.

3.4 Enrichment factors and analytical figures of merit

The enrichment factors (EF) were calculated for each sorbent by using the ratio between
the slope of the calibration curves obtained after extraction of 50 mL of standard mixtures
and the slope obtained by injecting directly the analytes into the chromatographic system
[30]. The concentration range used was 20–100 mg L1 for each compound in the extracted

Table 3. Breakthrough volume range (mL) obtained for the different sorbents
after extraction of volumes between 1–100 mL of P or 246TCP standard solutions
with a concentration of 500 mg L1.

Sorbent P 246TCP

Amberlite XAD-4 (commercial product) 40 5 V 5 50 75 5 V 5 100

Amberlite XAD-4 (grounded particles) 75 5 V 5 100 50 5 V 5 100
Macronet MN-200 25 5 V 5 50 V 4 100
Lichrolut EN V 4 75 V 4 100
 International Journal of Environmental Analytical Chemistry 893

250

200

150

EF
100

50

0
C

P
4N

2C

2N

3M

PC

C
O

6T
N

D
4C

24
D

24

24
46

Analyte

Figure 3. Enrichment factors (EF) for 50 mL of standard mixtures with concentrations in the range
6–80 mg L1 using the different sorbents (dotted bar, Amberlite XAD-4 (commercial product); white
bar, Amberlite XAD-4 (125–250 mm); black bar, Macronet MN-200; striped bar, Lichrolut EN).

mixtures and 500–8000 mg L1 in the standard mixtures used for direct injection. When
Lichrolut EN was used, the concentration of the compounds in the extracted mixtures
was adjusted for the range 6–30 mg L1. The results obtained in this experiment (Figure 3)
demonstrated that Lichrolut EN provided the higher values of enrichment factor (between
110 and 215), with a mean value of 176. The lower values of EF were obtained
for Amberlite XAD-4 with a range between 24 and 47 and an average value of 37. These
results could be explained for the differences between the two sorbents in the cross-linking
degree and consequently, in the surface area available for the interactions between
the analytes and the sorbent. However, for Amberlite XAD-4 with small particle size
(125–250 mm) the values of EF increased (values between 54 and 67) and were higher
than those obtained when using Macronet MN-200 (values between 29 and 57) that is
a polymer with a very high cross-linking degree. These results show that an enhancement
of the surface area by decreasing the particle size improved the capacity of the PS-DVB
sorbents with low cross-linking for the retention of these phenolic compounds, resulting
in performances comparable with sorbents with high cross-linking degree that by the
nature of their structure have higher surface areas.
Phenol and its primary derivatives listed as priority pollutants by the EPA (Table 1)
have distinct physical properties according to the different groups present in the aromatic
ring. Thus, the values obtained for EF were compared with the partition coefficient and
the molecular weight. In the case of Amberlite XAD-4, a positive correlation (R ¼ 0.867)
between the enrichment factor and the log Kow was observed (Figure 4). A linear
relationship was established: EF ¼ (10.96  7.24)  log Kow þ 12.0  17.0, where limits
of confidence are indicated for  ¼ 0.05, d.f. ¼ 5. Thus, the interval of confidence for
the slope value did not include the zero value and the slope value obtained was
significantly different from zero (tcalc ¼ 8.705, ttab ¼ 2.571,  ¼ 0.05, d.f. ¼ 5), providing
evidence about the linear relationship between these two variables [31]. This relationship
may be explained by the reversed-phase mechanism of interaction between the
phenolic compounds and the polymer, which promotes the adsorption of the more
hydrophobic compounds. A similar behaviour was found for the grounded
894 H.M. Oliveira et al.

80

60

EF

40

20

0
1 2 3 4
log k ow

Figure 4. Relation between enrichment factor (EF) and the partitioning coefficient (log Kow)
of phenolic compounds after SPE using Amberlite XAD-4 (commercial product (*) and grounded
particles (h)).

80 230

200
60
170
EF
EF

40 140

110
20
80

0 50
50 100 150 200 250 300 50 100 150 200 250 300
MW (u) MW (u)

Figure 5. Relation between enrichment factor (EF) and the molecular weight (MW) of phenolic
compounds obtained after SPE using the solid-phases Lichrolut EN (D) and Macronet MN-200 (h).

product: EF ¼ (6.64  4.81)  log Kow þ 45.4  11.3, R ¼ 0.846,  ¼ 0.05, d.f. ¼ 5. The slope
value was also significantly different from zero (tcalc ¼ 7.930, ttab ¼ 2.571,  ¼ 0.05,
d.f. ¼ 5). Nevertheless, for grounded Amberlite XAD-4, the values of EF were higher due
to the enhancement of the total surface area available for the extraction process.
Although the same mechanism of interaction was present, the behaviour reported for
Amberlite XAD-4 was not observed with the sorbents with high cross-linking degree
(Macronet MN-200 and Lichrolut EN, data not shown). Furthermore, for these sorbents,
a negative correlation between the EF and the molecular weight was found (Figure 5).
A linear relationship was established for Macronet MN-200: EF ¼ (0.146  0.105) 
MW þ 69.3  16.4, R ¼ 0.812,  ¼ 0.05, d.f. ¼ 6. Thus, the interval of confidence for the
Table 4. Analytical figures of merit obtained for each sorbent tested.

Amberlite XAD-4 (commercial) Amberlite XAD-4 (125–250 mm) Macronet Lichrolut

Tested Tested Tested Tested

conc./ Recovery conc./ Recovery conc./ Recovery LOD/ng conc./ Recovery
Compound LOD/ng mg L1 RSDa %b LOD/ng mg L1 RSDa %b LOD/ng mg L1 RSDa %b [23] mg L1 RSDa %b

24DNP 289 85.0 4.1 97.6  1.6 262 85.0 2.2 99.7  1.8 368 85.0 2.7 98.7  3.5 207 25.5 1.2 100.3  1.1
46DNOC 480 81.2 1.7 100.0  0.5 437 81.2 3.5 98.8  1.1 321 81.2 1.9 100.4  0.3 103 24.4 2.8 102.1  1.1
P 358 82.4 2.9 98.5  2.3 290 82.4 2.6 99.6  3.8 163 82.4 1.4 100.2  0.4 157 24.7 2.1 98.1  1.4
4NP 286 92.8 2.3 98.9  2.0 300 92.8 3.0 98.3  0.3 83 92.8 1.4 99.6  0.4 110 27.8 1.4 99.2  0.4
2CP 226 86.2 1.9 99.2  1.6 180 86.2 1.9 99.3  1.2 209 86.2 1.9 99.5  1.2 31 25.9 1.7 99.2  1.3
2NP 317 81.1 2.6 98.1  1.2 224 81.1 1.5 99.5  1.2 86 81.1 1.6 99.7  1.7 87 24.3 2.3 101.6  1.6
24DMP 293 84.1 1.3 99.5  0.1 297 84.1 2.9 98.4  1.6 171 84.1 1.6 99.1  0.5 30 25.2 1.6 99.1  0.7
4C3MP 292 79.9 1.4 99.5  0.3 224 79.9 1.2 99.8  1.5 98 79.9 1.4 99.7  0.4 18 24.0 2.0 100.9  3.4
24DCP 305 77.4 1.3 99.1  0.4 287 77.4 1.2 99.9  1.6 204 77.4 1.9 100.1  0.3 39 23.2 1.9 99.8  3.2
PCP 381 42.9 4.5 99.3  3.9 NA NA NA NA 227 42.9 1.9 100.6  2.4 66 12.9 2.2 100.2  5.0
246TCP 244 83.9 1.6 100.6  0.9 NA NA NA NA NA 83.9 1.6 102.7  2.7 55 25.2 5.1 NA

Notes: aCalculated using the statistics sy/x as estimate of standard deviation [33] (n ¼ 6 or n ¼ 12) divided by the tested concentration.
b
n ¼ 3.
NA ¼ not available.
International Journal of Environmental Analytical Chemistry
895
 896

Table 5. Analytical performance of on-line SPE chromatographic methods for determination of pollutant phenolics.

Sample Determination Organic

Methodology Working range/mg L1 LOD/mg L1 Sorbent volume/mL frequency/h1 solvent/mL Efluenta/mL Reference

LC-DAD 20–100 4.5–9.6 XAD-4 (commercial) 50 4 4.1–4.4c 9.5 Present work

LC-DAD 20–100 3.6–8.7 XAD (125–250 mm) 50 4 4.1–4.4c 9.5 Present work
LC-DAD 20–100 1.7–7.4 Macronet MN-200 50 4 4.1–4.4c 9.5 Present work
LC-DAD 6–30 0.4–4.1 Lichrolut EN 50 4 4.1–4.4c 9.5 Present work
LC-DAD 1.5–140 0.3–2 Lichrolut EN 25–100 4–10 4.1c 9.1 [23]
LC-ED 0.01–10 0.001–0.075 PLRP-S 1.0–4.0 2.6 16c 28 [33]
LC-MS 100–300 n. a. Severalb 100 2 3c 8 [34]
SFC-DAD 3–25 0.4–1.9 PLRP-s 20 1.5 10c 20 [35]
LC-UV 0.01–25 0.01–0.7 Lichrolut EN 100 1.6 5c þ 5d 12.5 [9]
LC-UV-FL 1.5–100 0.2–0.6 Modified PS-DVB 100 0.93–2 12c 24 [36]
H.M. Oliveira et al.

LC-UV-ED 0.05–20 0.035–0.07 Modified PS-DVB 20 2 12d 22 [10]

Notes: aExcluding sample volume.

b
Comparative study between C18 HD, PLRP-S, Hamilton-PRP-1, Hysphere GP, Hysphere SH and Oasis HLB.
c
Methanol.
d
Acetonitrile.
SFC, supercritical fluid chromatography; LC, liquid chromatography; DAD, diode-array detection; ED, electrochemical detection; MS, mass
spectrometry detection; UV, ultra-violet detection; FL, fluorimetric detection.
n.a. Not available.
 International Journal of Environmental Analytical Chemistry 897

slope value did not include the zero value and the slope value obtained was significantly
different from zero (tcalc ¼ 8.342, ttab ¼ 2.447,  ¼ 0.05, d.f. ¼ 6), providing evidence
about the linear relationship between these two variables [31]. For Lichrolut EN,
EF ¼ (0.562  0.251)  MW þ 267  40, R ¼ 0.913, and tcalc ¼ 13.411, ttab ¼ 2.447 for
 ¼ 0.05, d.f. ¼ 6. For these cases, the molecular size of the phenolic compound had
an important role on the extraction process because the high cross-linking between the
polymeric chains worked as a molecular sieve, promoting the sorption of the compounds
with low molecular size. These results are in agreement with recent observations from
Sychov et al. [32], indicating that the whole interior of the hypercross-linked polystyrene
particled is accessible to small analytes but not to larger molecules.
Concerning the analytical figures of merit (Table 4), the determination of LOD
values was based on the calibration curves established for the determination of EF and
they are given as mass of compound (present in 50 mL of sample), calculated from the
concentration obtained for the interception plus three times sy/x [31]. LOD values between
180 and 480 ng were obtained for Amberlite XAD-4, with mean values of 316 and 278 ng
for commercial and grounded sorbent, respectively. Lower values were obtained for
Macronet MN-200 (LOD between 83 and 368 ng, with mean value of 193 ng) and the
lowest values were obtained for Lichrolut EN (LOD between 18 and 207 ng, with mean
value of 82 ng). This was expected as the highest EF values were obtained for this sorbent.
The linear calibration range was 20–100 mg L–1 for a preconcentration volume
of 50 mL, except for Lichrolut EN. For this sorbent, the linear range attained was
6–30 mg L1 for the same sample volume. Repeatability was assessed through the statistic
sy/x, applied as an estimate of the standard deviation (n ¼ 6 or 12) [31]. Values lower than
4.5% were obtained for all sorbents tested (Table 4), accounting for the repeatable
conditions attained by the automation of the SPE procedure. Recovery studies were
performed using 50 mL of MilliQ water fortified with 80 mg L1 of each phenolic
compound (or 24 mg L1 for Lichrolut EN). Similar results were attained for all sorbents,
with recovery values between 97.6 and 102.7%. For Lichrolut EN further analytical
application was developed [23], providing statistically comparable results when certified
reference material was analysed. In fact, for samples RTC-QCI-032 and U-QCI-076
(Promochem, LGC Standards), the total phenolics content was in agreement with the
certified value [23]. Moreover no matrix effect was observed for mineral, tap and seawater
samples, with mean recovery values ranging from 89 to 103% for all analytes tested.
Compared to previously described on-line SPE methods for chromatographic
determination of phenolics [9,10,23,33–36], the sorbents studied here presented better or
similar performance (Table 5). In fact, organic solvent consumption was similar (4–5 mL)
and effluent production was lower (9.5 mL compared to 20–28 mL) in this work. The
method proposed by Wissiack et al. [34] provided better performance regarding these
aspects, but the concentration working range was higher and narrower.

4. Conclusions
In the present work, the utilisation of an automatic flow system provided repeatable
conditions for comparison of different SPE sorbents. For this, the implementation of
different flow directions for loading and elution operations was essential, because it
prevented the compaction of the sorbent bed and avoided the creation of preferable flow
paths. Compared to previous alternatives in this area, the multisyringe equipment was an
898 H.M. Oliveira et al.

advantageous choice as it was compatible with organic solvents because only glass syringes
and PTFE valves were in contact with the manipulated fluids. Moreover, the hyphenation
with the LC equipment through its injection valve was successful while the computer
control of the SPE protocol avoided unnecessary reagent (conditioning solution, eluent)
consumption during the chromatographic run.
Concerning the performance of the sorbents, higher breakthrough volumes were
obtained for Lichrolut EN for both model analytes studied (P and 246TCP). Higher values
were also obtained for Lichrolut EN, followed by Amberlite XAD-4 (grounded particles),
Macronet MN-200 and Amberlite XAD-4 (commercial particles), regarding the enrich-
ment factors obtained under the same experimental conditions. As a consequence, lower
values of LOD were also attained when applying Lichrolut EN. Nevertheless, repeatability
and recovery values were similar for all sorbents tested. These results indicate Lichrolut
EN as the best choice for analytical applications because lower concentrations can be
determined by using this sorbent, fostering the determination in samples at low ng mL1
levels using conventional HPLC-DAD technique [23].
Concerning the molecular interactions between the analytes and the sorbent, it was
observed a positive correlation between the EF values and the hydrophobic character
of the phenolic compound (expressed as log Kow) for Amberlite XAD-4, justified by the
enhanced retention of more hydrophobic molecules due to the prevalence of the reversed-
phase mechanism of interaction between the analytes and this sorbent. For the sorbents
with higher cross-linking degree, this was not observed. In fact, a negative correlation
between the EF values and the molecular weight of the analyte was verified, indicating
that the structure of these sorbents may act like a molecular sieve, granting or restricting
access to inner surfaces according to the analyte size. This fact may have consequences not
only in the analytical application of these sorbents but also on their application
for removal of phenolic pollutants in effluents. In both situations, larger molecules will be
less retained despite the enhancement of surface area brought by the higher cross-linking
degree.

Acknowledgements
Hugo M. Oliveira thanks Fundação para a Ciência e Tecnologia (FCT) and FSE (III Quadro
Comunitário) for the grant SFRH/BD/22494/2005. We acknowledge Neoquı́mica for the gift of
Macronet MN-200 sorbent.

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